The goal of this proposal is to examine the mechanism of the vascular myogenic response and its role in regulating microvascular pressure and flow. This will be approached with studies at three levels of organization: microvascular networks, isolated perfused microvessels, and single vascular smooth muscle cells. The research will address three specific objectives: 1) The first objective is to assess the role of the myogenic response in microvascular networks in vivo. We will test the hypothesis that longitudinal gradients in myogenic responsiveness exist within arteriolar networks. Arterioles of the hamster cheek pouch and cremaster muscle will be studied using intravital microscopic techniques for measuring pressure, diameter and flow. Arteriolar pressure will be manipulated using box and occlusion techniques in order to elicit arteriolar myogenic responses. We will also test the hypothesis that myogenic and flow-mediated mechanisms interact additively and competitively in vivo. Blockers of endothelial-dependent, flow-mediated dilation will be used to assess the contribution of this mechanism. 2) The second objective is to determine the mechanism and function of the myogenic response in single arterioles. Isolated, cannulated arteriolar segments from hamster cheek pouch and cremaster muscle will be studied in vitro to determine how myogenic responsiveness varies with vessel size. These studies will be performed in parallel with the in vivo studies above and used to help interpret in vivo experiments. The interaction of myogenic and endothelial-dependent mechanisms will be examined by varying pressure and flow independently. Double vessel preparations (two vessels perfused in series) will be used to test the hypothesis that flow-mediated responses involve the release of transferable dilator and constrictor factors from the endothelium. 3) The third objective is to examine the cellular mechanism of the myogenic response. Vascular smooth muscle (VSM) cells will be enzymatically dispersed from arterioles and studied using whole-cell and single-channel patch-clamp techniques. We will test the hypothesis that stretch-induced contraction of VSM requires Ca2+ entry via stretch- activated channels and voltage-gated calcium channels. The direct effect of flow/shear-stress on VSM cells and the associated ionic mechanisms will also be studied. The intracellular fluorescent probe Indo-1 will be used to quantitate changes in cytoplasmic [Ca2+] as a function of single vascular smooth muscle cell stretch. The results of these experiments will provide new insights into the mechanism and function of the myogenic response at the network, single vessel, and cellular levels.